The primary goal is to determine the mechanism by which lipid containing viruses assemble and bud from the cell membrane. This project deals with the main structural component of influenza virus, the Ml protein and has two purposes. First, the exact association of Ml with the cell lipid and the interactions of Ml with the other viral proteins that leads to virus formation will be determined. Second, the importance of phosphorylation of Ml in virus assembly will be investigated by establishing the quantify of phosphate, the site of phosphorylation and determining if there is conservation of phosphorylation of the Ml in groups B and C influenza viruses. These studies will involve the use of chemical cross-linking reagents of varying lengths and varying types, isolation of components by immunoprecipitation, isolation of subcellular fractions on gradients and immunofluorescent techniques on whole cells. Assays will be used for quantitating phosphate. Proteolytic enzymes and chemical agents will be used to cleave Ml. Chromatographic procedures will be used to separate peptides which will be analyzed for amino acid content and sequence. A recombinant virus containing a clone of Ml will be used to study the pathway and association of Ml in the cell and its phosphorylation in the absence of other proteins.